ABSTRACT There exist fundamental gaps in our understanding of the transcriptional regulatory pathways through which microbiota and inflammation alter gene expression in the intestinal epithelium, and how those pathways promote and might predict intestinal homeostasis. Our long-term goal is to understand the evolutionarily-conserved mechanisms underlying host-microbe interactions in the intestine and how they contribute to human diseases. The overall objectives of this project are to identify conserved transcriptional regulatory pathways mediating intestinal epithelial cell (IEC) responses to microbiota and inflammation, and determine if their activity can be used as phenotypic indicators in human inflammatory bowel disease (IBD). Our preliminary studies in human, mouse, and zebrafish IECs have uncovered striking conservation of transcriptional signatures and regulatory mechanisms, predicting central conserved roles for transcription factors (TF) implicated in human IBD including HNF4A and ELF3. We recently made the key discovery that HNF4A is a novel mediator of IEC transcriptional responses to microbiota in zebrafish and mice. Mechanistic studies in zebrafish and mice revealed that HNF4A is a positive regulator of microbially-suppressed genes, and that microbiota broadly suppress HNF4A activity in IECs. Further, our results show that intestinal suppression of HNF4A target genes is a prevalent feature of human, mouse, and zebrafish models of IBD, and indicate that HNF4A constrains inflammatory responses to microbiota and suppresses a conserved IBD-like gene expression signature. Finally, we identified DNA enhancer elements in mouse IECs that are regulated by microbiota colonization, and leverage those results to implicate ELF3 as a potential integrator of inflammatory and microbial signals in IECs. We will test the central hypothesis that microbiota promote intestinal inflammation by coordinately suppressing anti-inflammatory HNF4A activity and activating ELF3 and other pro-inflammatory transcriptional pathways. In Specific Aim 1, we will identify host signaling mechanisms mediating microbial suppression of HNF4A activity in IECs. In Specific Aim 2, we will define the roles of ELF3 and broader transcriptional pathways in IEC responses to microbiota and inflammation, and test if IBD phenotypes are associated with distinct signatures of ELF3 and HNF4A activity. The expected outcomes will vertically advance the field in several ways. First, they will generate a foundational mechanistic understanding of how gut microbes regulate the activity of HNF4A and ELF3, conserved TFs with critical roles in intestinal inflammation. Second, they will provide much-needed insights into the transcriptional regulatory networks utilized by IECs to integrate microbial and inflammatory stimuli, and how those networks are deranged in human IBD. These outcomes are expected to have a significant impact because they will vertically advance our understanding of the IEC transcriptional programs that integrate responses to microbiota and inflammation, which can be expected to lead to new diagnostic, prognostic, and therapeutic approaches for human IBD.